CN112480122B

CN112480122B - Tetrahydroimidazo [1,2-a ] pyrazine compound, composition, preparation method and application thereof

Info

Publication number: CN112480122B
Application number: CN202011327191.4A
Authority: CN
Inventors: 熊小峰; 张小雷; 葛阳; 石硕; 邓俊杰; 刘璐; 陈新滋
Original assignee: Sun Yat Sen University
Current assignee: Sun Yat Sen University
Priority date: 2020-11-24
Filing date: 2020-11-24
Publication date: 2022-03-22
Anticipated expiration: 2040-11-24
Also published as: CN112480122A

Abstract

The invention discloses a tetrahydroimidazo [1,2-a ]]Pyrazine compounds, compositions, and methods of preparation and use thereof. The compound has a structure shown in formula (I), has G protein resistant activity superior to BIM-46174, and has no toxicity to normal cells; and the preparation is simple and the cost is low.

Description

Tetrahydroimidazo [1,2-a ] pyrazine compound, composition, preparation method and application thereof

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to a tetrahydroimidazo [1,2-a ] pyrazine compound, a composition, a preparation method and an application thereof.

Background

G protein-coupled receptors (GPCRs) are the largest family of cell surface receptors, over 800 known GPCRs have been identified in the human genome, and GPCRs are widely expressed in vivo and are involved in a large number of physiological and pathological processes. GPCRs mediate a variety of physiological functions by coupling to the intracellular heterotrimeric guanine nucleotide binding protein (G protein), passing a number of extracellular signals through the cell membrane. GPCRs are currently the most common class of drug targets, with about 25% of drugs approved for marketing by the FDA in the united states targeting GPCRs, suggesting that GPCR-G protein signaling pathways play an important role in drug development.

The heterotrimeric G protein is used as a molecular switch in the signal transduction process of a G protein-coupled receptor (GPCR), transmits information from the outside of the cell to the inside of the cell, and regulates the growth and development process of organisms through a series of cascade reactions. The G protein is located inside the cell membrane and is a heterotrimer composed of α, β, and γ subunits, with different subunits performing different biological functions. When a GPCR is activated, a signal can be transmitted to the downstream G protein through a conformational change, resulting in activation of the G protein. When the G protein is in the inactive state, the G α subunit binds Guanosine Diphosphate (GDP) and forms a stable trimer with the dimeric G β γ subunit. When the G protein is activated, G.alpha.changes from the inactivated state G.alpha.GDP bound to GDP to the activated state G.alpha.GTP bound to Guanosine Triphosphate (GTP), and then G.alpha.and G.beta.gamma.are separated and the respective signaling is initiated. The activated G protein will initiate further signaling, activating a number of "second messenger" systems and initiating a range of important physiological activities. The signal is terminated by the G protein signal regulatory protein (RGS) binding to G.alpha.subunit to play the role of GAP (GTPase activating protein) to hydrolyze GTP into GDP, and then the G.alpha.subunit binding to GDP is recombined with G.beta.gamma subunit to form G.alpha.gamma heterotrimer, and the G protein returns to the inactive state.

G proteins can be classified as G.alpha.based on the homology of the G protein's alpha subunit_q/11、Gα_i、Gα_sAnd G.alpha._12/13Four subfamilies. G alpha_sCan increase the catalytic activity of downstream Adenosine Cyclase (AC) and lead to the increase of the cyclic adenosine monophosphate (cAMP) level of a second messenger, and then activate Protein Kinase A (PKA) to trigger a series of downstream signal transmission processes. And G alpha_sIn contrast, G.alpha._iThe activation of (b) inhibits the activity of AC, resulting in a decrease in cAMP levels. G alpha_12/13Is capable of activating small GTPases. G alpha_q/11Is capable of activating phospholipase C beta (PLC beta), which is capable of hydrolyzing 4, 5-diphosphatidyinositol (PIP2) and producing two distinct "second messengers," Diacylglycerol (DAG) and inositol triphosphate (IP3), wherein DAG is capable of activating Protein Kinase C (PKC), and IP3 is capable of activating ligand-gated Ca on the endoplasmic reticulum²⁺Ion channels and promote Ca²⁺Ion release to the cytoplasm, and the generation of DAG and IP3 will trigger physiological phenomena such as platelet aggregation, smooth muscle contraction, exocytosis and the like. In addition to normal physiological processes, G protein is an important mediator in a variety of tumors (e.g., uveal melanoma, prostate)Cancer, pancreatic cancer, leukemia, glioblastoma) and inflammation (e.g., asthma). The G protein has become one of the important targets for research and development of antitumor drugs.

The G protein small molecule inhibitor has good prospect for treating malignant tumor and inflammatory diseases. FR900359 and YM-254890 as highly selective Ga_q/11Inhibitors, IC thereof₅₀32nM and 95nM, respectively, have been used as tool molecules for studying uveal melanoma and asthma (Onken, m., et al. sci. signal.,2018,11, eaao 6852; Matthey, m., et al. sci. trans. med.,2017,9, eaag 2288), but there is a great limitation in disease treatment due to great difficulty in synthesis. EC for inhibition of G protein by Suramin₅₀240nM (Freismuth, M., et al. mol. Pharmacol.,1996,49, 602-611.). BIM-46174 is a G protein inhibitor (US7034024B1, WO0134203A1, EP1430934A1) developed by French SOD CONSEILS RECH APPLIC, and its dimer BIM-46187 is directed to G.alpha._q/11The selectivity of (Ayou ub, M., et al J. biol. chem.,2009,284, 29136-29145; Schmitz, A., et al chem. biol.,2014,21, 890-902) is high. Other compounds, such as 0990(Appleton, k., et al bioorg.med.chem.,2014,22,3423-_iProtein selectivity; m119 and Gallein are highly selective G.beta.gamma.inhibitors, the IC of which₅₀Is 200nM (Bonacci, T., et al. science,2006,312, 443-. The small molecule inhibitor has the problems of high toxicity, poor structural stability, low antitumor activity and the like, so that the subsequent development and research of the small molecule inhibitor are limited.

In view of the urgent need for cancer treatment, there is a need in the art to develop new drugs with better efficacy.

Disclosure of Invention

The invention aims to overcome the defects in the prior art and provides a tetrahydroimidazo [1,2-a ] pyrazine compound or pharmaceutically acceptable salt thereof, wherein the compound has good antitumor activity.

The present invention also provides a pharmaceutical composition of the above tetrahydroimidazo [1,2-a ] pyrazines compound or a pharmaceutically acceptable salt thereof.

The invention further aims to provide the tetrahydroimidazo [1,2-a ] pyrazine compound or pharmaceutically acceptable salt thereof, or application of the composition.

The purpose of the invention is realized by the following technical scheme: a tetrahydroimidazo [1,2-a ] pyrazine compound or a pharmaceutically acceptable salt thereof, wherein the chemical structure of the tetrahydroimidazo [1,2-a ] pyrazine compound is represented by the formula (I):

wherein the content of the first and second substances,

x is selected from carbonyl or sulfonyl;

l is selected from-CH-or hydrogen;

R₁selected from hydrogen or methyl;

R₂selected from hydrogen, methyl, ethyl, isopropyl, hydroxyl, mercapto, carboxyl, cyano,

R₃Selected from methyl, amino, hydroxyl,

R₄Selected from methyl, isopropyl, and isopropyl,

As a specific embodiment of the invention, the compound represented by the general formula (I) has a structure shown in formulas I-1 to I-36:

the structural compound is a tetrahydroimidazo [1,2-a ] pyrazine compound, and the screening of the antitumor activity of the invention shows that part of the compounds have stronger capability of inhibiting the proliferation of uveal melanoma cells (92.1 and MP41), and part of the compounds show better anti-G protein activity than BIM-46174. As a molecule with novel structure, the compound has the potential of being developed into a novel high-efficiency G protein inhibitor, and has great application value in treating related tumor diseases, particularly uveal melanoma, prostatic cancer, acute myeloid leukemia, pancreatic cancer or glioblastoma.

The structures represented by the foregoing I-1 to I-36 have the following names, respectively:

(I-1) (S) -3-amino-4- ((S) -8- (cyclohexylmethyl) -2-phenyl-5, 6-dihydroimidazo [1,2-a ] piperazin-7 (8H) -yl) -4-oxobutanenitrile;

(I-2) (S) -2-amino-1- ((S) -8- (cyclohexylmethyl) -2-phenyl-5, 6-dihydroimidazo [1,2-a ] piperazin-7 (8H) -yl) -3-methylbutan-1-one;

(I-3) (S) -2-amino-1- ((S) -8- (cyclohexylmethyl) -2-phenyl-5, 6-dihydroimidazo [1,2-a ] piperazin-7 (8H) -yl) -3- (4-hydroxyphenyl) propan-1-one;

(I-4) (S) -2-amino-1- ((S) -8- (cyclohexylmethyl) -2-phenyl-5, 6-dihydroimidazo [1,2-a ] piperazin-7 (8H) -yl) -3- (1H-imidazol-4-yl) propan-1-one;

(I-5) (S) -2-amino-1- ((S) -8- (cyclohexylmethyl) -2-phenyl-5, 6-dihydroimidazo [1,2-a ] piperazin-7 (8H) -yl) propan-1-one;

(I-6) (S) -2-amino-1- ((S) -8- (cyclohexylmethyl) -2-phenyl-5, 6-dihydroimidazo [1,2-a ] piperazin-7 (8H) -yl) -4-methylpentane-1-one;

(I-7) (S) -2-amino-1- ((S) -8- (cyclohexylmethyl) -2-phenyl-5, 6-dihydroimidazo [1,2-a ] piperazin-7 (8H) -yl) -3-phenylpropan-1-one;

(I-8) (S) -3-amino-4- ((S) -8- (cyclohexylmethyl) -2-phenyl-5, 6-dihydroimidazo [1,2-a ] piperazin-7 (8H) -yl) -4-oxobutanoic acid;

(I-9)1- ((S) -4-amino-5- ((S) -8- (cyclohexylmethyl) -2-phenyl-5, 6-dihydroimidazo [1,2-a ] piperazin-7 (8H) -yl) -5-oxopentyl) guanidine;

(I-10) (2S,3R) -2-amino-1- ((S) -8- (cyclohexylmethyl) -2-phenyl-5, 6-dihydroimidazo [1,2-a ] piperazin-7 (8H) -yl) -3-methylpentane-1-one;

(I-11) (S) -4-amino-5- ((S) -8- (cyclohexylmethyl) -2-phenyl-5, 6-dihydroimidazo [1,2-a ] piperazin-7 (8H) -yl) -5-oxopentanoic acid;

(I-12) (S) -3-amino-4- ((S) -8- (cyclohexylmethyl) -2-phenyl-5, 6-dihydroimidazo [1,2-a ] piperazin-7 (8H) -yl) -4-oxobutanamide;

(I-13) (2S,3R) -2-amino-1- ((S) -8- (cyclohexylmethyl) -2-phenyl-5, 6-dihydroimidazo [1,2-a ] piperazin-7 (8H) -yl) -3-hydroxybutan-1-one;

(I-14) (S) -2, 6-diamino-1- ((S) -8- (cyclohexylmethyl) -2-phenyl-5, 6-dihydroimidazo [1,2-a ] piperazin-7 (8H) -yl) hexan-1-one;

(I-15) (S) -4-amino-5- ((S) -8- (cyclohexylmethyl) -2-phenyl-5, 6-dihydroimidazo [1,2-a ] piperazin-7 (8H) -yl) -5-oxovaleramide;

(I-16) (S) -2-amino-1- ((S) -8- (cyclohexylmethyl) -2-phenyl-5, 6-dihydroimidazo [1,2-a ] piperazin-7 (8H) -yl) -3- (1H-indol-3-yl) propan-1-one;

(I-17) (S) -2-amino-3-cyclohexyl-1- ((S) -8- (cyclohexylmethyl) -2-phenyl-5, 6-dihydroimidazo [1,2-a ] piperazin-7 (8H) -yl) propan-1-one;

(I-18) (S) -1- ((S) -8- (cyclohexylmethyl) -2-phenyl-5, 6-dihydroimidazo [1,2-a ] piperazin-7 (8H) -yl) -3-mercapto-2-methylaminopropane-1-one;

(I-19) (S) -1- ((S) -8- (cyclohexylmethyl) -2-phenyl-5, 6-dihydroimidazo [1,2-a ] piperazin-7 (8H) -yl) -2- (dimethylamino) -3-mercaptopropan-1-one;

(I-20) (S) -1- ((S) -8- (cyclohexylmethyl) -2-phenyl-5, 6-dihydroimidazo [1,2-a ] piperazin-7 (8H) -yl) -2- (diethylamino) -3-mercaptopropan-1-one;

(I-21) (S) -1- (8- (cyclohexylmethyl) -2-phenyl-5, 6-dihydroimidazo [1,2-a ] piperazin-7 (8H) -yl) -2-methylpropan-1-one;

(I-22)1- ((S) -8- (cyclohexylmethyl) -2-phenyl-5, 6-dihydroimidazo [1,2-a ] piperazin-7 (8H) -yl) -2-methylbutan-1-one;

(I-23) (2S,3R) -2-amino-1- ((S) -8-benzyl-2-phenyl-5, 6-dihydroimidazo [1,2-a ] piperazin-7 (8H) -yl) -3-methylpentane-1-one;

(I-24) (S) -2-amino-1- ((R) -8- (cyclohexylmethyl) -2-phenyl-5, 6-dihydroimidazo [1,2-a ] piperazin-7 (8H) -yl) -3- (4-hydroxyphenyl) propan-1-one;

(I-25) (S) -2-amino-1- ((R) -8-isobutyl-2-phenyl-5, 6-dihydroimidazo [1,2-a ] piperazin-7 (8H) -yl) -3-mercaptopropan-1-one;

(I-26) (S) -2-amino-1- ((S) -8- ((R) -sec-butyl) -2-phenyl-5, 6-dihydroimidazo [1,2-a ] piperazin-7 (8H) -yl) -3-mercaptopropan-1-one;

(I-27) (S) -2-amino-1- ((S) -8-isopropyl-2-phenyl-5, 6-dihydroimidazo [1,2-a ] piperazin-7 (8H) -yl) -3-mercaptopropan-1-one;

(I-28) (S) -2-amino-3-mercapto-1- ((S) -8-methyl-2-phenyl-5, 6-dihydroimidazo [1,2-a ] piperazin-7 (8H) -yl) propan-1-one;

(I-29) (S) -8- (cyclohexylmethyl) -7- (isopropylsulfonyl) -2-phenyl-5, 6,7, 8-tetrahydroimidazo [1,2-a ] piperazine;

(I-30) (S) -2-amino-1- (8- (cyclohexylmethyl) -2-phenyl-5, 6-dihydroimidazo [1,2-a ] piperazin-7 (8H) -yl) ethan-1-one;

(I-31) (S) -8- (cyclohexylmethyl) -7- (ethylsulfonyl) -2-phenyl-5, 6,7, 8-tetrahydroimidazo [1,2-a ] piperazine;

(I-32) (S) -8-benzyl-7- (isopropylsulfonyl) -2-phenyl-5, 6,7, 8-tetrahydroimidazo [1,2-a ] piperazine;

(I-33) (S) -2, 6-diamino-1- ((R) -8-isobutyl-2-phenyl-5, 6-dihydroimidazo [1,2-a ] piperazin-7 (8H) -yl) hexan-1-one;

(I-34) (S) -1- ((S) -8- (cyclohexylmethyl) -2-phenyl-5, 6-dihydroimidazo [1,2-a ] piperazin-7 (8H) -yl) -2-hydroxypropan-1-one;

(I-35) (R) -1- (8-isobutyl-2-phenyl-5, 6-dihydroimidazo [1,2-a ] piperazin-7 (8H) -yl) -2-methylpropan-1-one;

(I-36) (2S,3R) -2-amino-3-methyl-1- ((R) -8-methyl-2-phenyl-5, 6-dihydroimidazo [1,2-a ] piperazin-7 (8H) -yl) pentan-1-one.

The preparation method of the tetrahydroimidazo [1,2-a ] pyrazine compound comprises the following steps:

(1) dissolving a compound 1 with a structural formula shown as a formula 1 and an acid-binding agent A in an organic solvent A, adding a compound 2 with a structural formula shown as a formula 2 for reaction, and removing the organic solvent A after the reaction is finished; adding an organic solvent B for resuspension, adding ammonium acetate, carrying out reflux reaction, and removing the organic solvent B after the reaction is finished; adding water and an extracting agent for extraction, and taking an extracting agent layer; washing, dewatering and concentrating the obtained extractant layer to obtain a compound 3;

(2) dissolving the compound 3 and an acid-binding agent C in an organic solvent C, adding ethyl bromoacetate, cooling after reaction, and adding water to separate out a compound 4;

(3) dispersing compound 4 and catalyst D in organic solvent D, sealing, and charging H₂Reacting, filtering, concentrating the obtained filtrate, and purifying to obtain a compound 5;

(4) dispersing the compound 5 and borane tetrahydrofuran complex solution in an organic solvent E, sealing, introducing argon, carrying out a first reflux reaction, and removing the organic solvent E after TLC monitoring reaction is finished; adding organic solvent F, refluxing for the second time, concentrating, and purifying to obtain compound 6

(5) Preparation of tetrahydroimidazo [1,2-a ] pyrazines by step (a) or by step (B):

(A) dissolving the compound 7 in an organic solvent G, and adding diisopropylcarbodiimide to carry out a first reaction; then adding a compound 6, carrying out a second reaction, and after the TLC monitoring reaction is finished, concentrating and purifying; adding CF to the obtained product under inert atmosphere₃COOH and triisopropylsilane are reacted for the third time to obtain the tetrahydroimidazol [1,2-a ] with the structural formula shown in the formula (I)]Pyrazine compounds;

x is selected from carbonyl or sulfonyl; l is selected from-CH-or hydrogen; r₁Selected from hydrogen or methyl;

R₃Selected from methyl, amino, hydroxyl,

R₄Selected from methyl, isopropyl, and isopropyl,

(B) Dissolving the compound 8 and triethylamine in an organic solvent H, adding the compound 6, reacting, monitoring by TLC, concentrating and purifying to obtain the tetrahydroimidazo [1,2-a ] pyrazine compound with the structural formula shown in the formula (I).

The preparation method of the pharmaceutically acceptable salt is to react the tetrahydroimidazo [1,2-a ] pyrazine compound with organic acid or inorganic acid to obtain the pharmaceutically acceptable salt.

The compound 1 described in the step (1) is preferably (S) -2- (((benzyloxy) carbonyl) amino) -3-cyclohexylpropionic acid, N-benzyloxycarbonyl-L-phenylalanine, (R) -2- (((benzyloxy) carbonyl) amino) -3-cyclohexylpropionic acid, N-benzyloxycarbonyl-D-leucine, N-benzyloxycarbonyl-L-isoleucine, N-benzyloxycarbonyl-L-valine, N-benzyloxycarbonyl-L-alanine, N-benzyloxycarbonyl-D-leucine, N-benzyloxycarbonyl-D-alanine.

The acid-binding agent A in the step (1) is preferably Cs₂CO₃。

The dosage of the acid-binding agent A is preferably the same molar weight as that of the compound 1.

The organic solvent A in step (1) is preferably DMF.

The organic solvent A is a reaction medium and is used for dissolving reaction substances; preferably, 30mL of organic solvent A is used per 15.3mmol of compound 1.

The reaction temperature in step (1) is room temperature.

The room temperature is 10-40 ℃; preferably 20-30 ℃; more preferably 24 to 28 ℃.

The reaction time in the step (1) is 10-30 min; preferably 20 min.

The organic solvent A is removed in the step (1) preferably by rotary evaporation.

The organic solvent B in the step (1) is preferably toluene.

The dosage of the ammonium acetate is preferably 8 to 10 times of the 1 molar weight of the compound; more preferably 9 times.

The temperature of the reflux reaction described in the step (1) depends on the boiling point of the organic solvent B used.

The time of the reflux reaction in the step (1) is preferably 1-3 h; preferably for 2 hours.

The organic solvent B is removed in the step (1) preferably by rotary evaporation.

The water in the step (1) is selectively added, and the use amount of the water is to increase the volume of the reactant, so that an extractant layer is obtained. If the extraction is repeated, water is added at the first extraction.

The extractant in the step (1) is preferably ethyl acetate.

The number of extractions described in step (1) is preferably 3.

The washing in step (1) is preferably carried out with a saturated brine.

The water removal in the step (1) is preferably performed by using anhydrous sodium sulfate.

The concentration in the step (1) is preferably performed by a reduced pressure rotary evaporation method.

In the step (2)The acid-binding agent C is preferably K₂CO₃。

The dosage of the acid-binding agent C in the step (2) is preferably 1-2 times of the molar weight of the compound 3; more preferably 1.5 times.

The organic solvent C in step (2) is preferably DMF.

The organic solvent C in the step (2) is used for forming a solution reaction system and is a reaction medium; the amount of the compound is preferably less than 28.9mmol of the compound 3 and 50mL of the organic solvent C.

The reaction temperature in the step (2) is preferably 40-60 ℃; more preferably 50 deg.c.

The reaction time in the step (2) is preferably 1-3 h; more preferably 2 h.

The catalyst D described in step (3) is preferably Pd/C having a dry content of 10%.

The organic solvent D in the step (3) is preferably methanol.

The dosage of the organic solvent D is preferably 15mL of methanol per 7.36mmol of the compound 4.

The temperature of the reaction in the step (3) is room temperature.

The reaction time in the step (3) is preferably 6-10 h; more preferably 8 h.

The filtration described in step (3) is preferably by filtration through celite.

The compound 6 and the borane tetrahydrofuran complex in the step (4) are preferably mixed in a molar ratio of 1: 5-7; more preferably, the molar ratio of 1: 6 to prepare a mixture ratio.

The organic solvent E in the step (4) is preferably tetrahydrofuran.

The dosage of the organic solvent E is preferably 20mL per 9.7mmol of the compound 6.

The time for the first reflux reaction described in step (4) is preferably 8 hours.

The removal of the organic solvent E in step (4) is preferably carried out by rotary evaporation.

The time for the second reflux reaction in step (4) is preferably 2 hours.

The compound 7 in the step (A) is preferably N-t-butoxycarbonyl-L-asparagine, Boc-L-valine, Boc-O-t-butyl-L-tyrosine, N-t-butoxycarbonyl-N' -trityl-L-histidine, N-t-butoxycarbonyl-L-alanine, N-t-butoxycarbonyl-L-leucine, N-t-butoxycarbonyl-L-phenylalanine, t-butoxycarbonyl-L-aspartic acid-4-t-butyl ester, tri-t-butoxycarbonylarginine, N-t-butoxycarbonyl-L-isoleucine, N-t-butoxycarbonyl-L-glutamic acid-5-t-butyl ester, t-butoxycarbonyl-N-beta-trityl-L-asparagine, Boc-L-histidine, N-t-butyloxycarbonyl-L-alanine, N-t-butyloxycarbonyl-L-isoleucine, N-t-butoxycarbonyl-L-glutamic acid-5-t-butyl ester, N-beta-trityl-L-asparagine, or L-histidine, N-tert-butyloxycarbonyl-O-tert-butyl-L-threonine, (S) -2, 6-di-tert-butyloxycarbonylaminohexanoic acid, N-tert-butyloxycarbonyl-N '-trityl-L-glutamine, N-tert-butyloxycarbonyl-N' -tert-butyloxycarbonyl-L-tryptophan, (S) -2- ((tert-butoxycarbonyl) amino) -3-cyclohexylpropionic acid, N-Boc-N-methyl-S-trityl-L-cysteine, N-dimethyl-S-trityl-L-cysteine, N-diethyl-S-trityl-L-cysteine, isobutyric acid, and mixtures thereof, (S) -2-methylbutyric acid, N-tert-butoxycarbonyl-L-isoleucine, Boc-O-tert-butyl-L-tyrosine, N-tert-butoxycarbonyl-S-trityl-L-cysteine, N-tert-butoxycarbonylglycine, (S) -2, 6-di-tert-butoxycarbonylaminocaproic acid or O-tert-butoxycarbonyl-L-lactic acid.

The organic solvent G in step (A) is preferably dichloromethane.

The amount of the organic solvent G used in the step (A) is preferably 2mL per 0.43mmol of the compound (7).

The diisopropylcarbodiimide used in step (a) is used in excess relative to compound 7; preferably according to compound 7: diisopropylcarbodiimide in a molar ratio of 1: 1.01-2 proportion calculation; more preferably according to compound 7: diisopropylcarbodiimide in a molar ratio of 1: 1.5 calculating the mixture ratio.

The time for the first reaction described in step (A) is preferably 30 min.

The compound 6 is used in an excess amount relative to the compound 7 in the step (A); preferably according to compound 7: compound 6 in a molar ratio of 1: 1.01-1.2 proportion calculation; more preferably according to compound 7: compound 6 in a molar ratio of 1: 1.1 calculating the mixture ratio.

The time of the second reaction in the step (A) is preferably 6-16 h.

CF described in step (A)₃The amount of COOH used is preferably 0.8 to 1.2mL of CF per 7mmol of the compound₃Calculating COOH; 1mL of CF per 0.43mmol of compound 7₃COOH calculation.

The triisopropylsilane is used in an excess amount relative to compound 7 in step (A); preferably according to compound 7: triisopropylsilane in a molar ratio of 1: 1.01-1.2 proportion calculation; more preferably according to compound 7: triisopropylsilane in a molar ratio of 1: 1.1 calculating the mixture ratio.

The time for the third reaction described in step (A) is preferably 2 h.

The step (A) also comprises the steps of filtering, concentrating and purifying the reaction liquid obtained by the third reaction.

The purification is preferably by semi-preparative liquid phase purification.

The compound 8 described in step (B) is preferably isopropyl sulfonyl chloride or ethyl sulfonyl chloride.

The dosage of the triethylamine in the step (B) is 2-2.1 times of the molar weight of the compound 8.

The organic solvent H used in step (B) is preferably dichloromethane.

The amount of the organic solvent G used in the step (B) is preferably 2mL of the organic solvent H per 0.7mmol of the compound 8.

The molar amount of the compound 6 in the step (B) is equivalent to the molar amount of the compound 8.

The reaction in the step (B) is preferably carried out for 6-16 h at 35-45 ℃; more preferably, the reaction is carried out for 6 to 16 hours at 40 ℃.

The specific steps of purification described in steps (3), (4) and (5) are preferably as follows: performing silica gel column chromatography, and spin-drying.

The silica gel is preferably silica gel with the particle size of 200 and 300 meshes.

In another aspect, the present invention provides a pharmaceutical composition comprising an effective amount of a compound represented by the general formula (i) or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

The drug carrier is preferably liposome or nanoparticle.

The salts of the compounds of formula (I) are preferably pharmaceutically acceptable salts, because of their potential use in medicine. The compounds of the present invention are bases, wherein the desired salt form can be prepared by suitable methods known in the art, including treatment of the free base with a mineral acid, such as hydrochloric acid, hydrobromic acid, sulfuric acid, sulfurous acid, pyrosulfuric acid, nitric acid, phosphoric acid, and the like; or treating the free base with an organic acid, such as acetic acid, trifluoroacetic acid, maleic acid, succinic acid, mandelic acid, fumaric acid, malonic acid, pyruvic acid, oxalic acid, glycolic acid, salicylic acid, pyranosidyl acid (pyranosidy1 acid), such as glucuronic acid or galacturonic acid, an alpha-hydroxy acid, such as citric acid or tartaric acid, an amino acid, such as aspartic acid or glutamic acid, an aromatic acid, such as benzoic acid or cinnamic acid, a sulfonic acid, such as p-toluenesulfonic acid, methanesulfonic acid, ethanesulfonic acid, and the like. Examples of pharmaceutically acceptable salts include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, chloride, bromide, iodide, acetate, propionate, caprate, caprylate, acrylate, formate, isobutyrate, hexanoate, heptanoate, propionate, oxalate, malonate, benzoates, chlorobenzoates, methylbenzoates, dinitrobenzoates, hydroxybenzoates, methoxybenzoates, phthalates, phenylacetates, phenylpropionates, phenylbutyrates (phenylbutyrates), citrates, lactates, gamma-hydroxybutyrates, hydroxyacetates, tartrates, mandelates and sulfonates, such as xylenesulfonates, methanesulfonates, propanesulfonates, naphthalene-1-sulfonate and naphthalene-2-sulfonate.

The pharmaceutical compositions of the invention will generally contain one compound of the invention. However, in some embodiments, the pharmaceutical compositions of the invention contain more than one compound of the invention. In addition, the pharmaceutical compositions of the present invention may optionally further comprise one or more other pharmaceutically active compounds.

The invention also provides the tetrahydroimidazo [1,2-a ] pyrazine compound or a pharmaceutically acceptable carrier thereof, and application of the pharmaceutical composition in inhibiting tumor proliferation by inhibiting G protein. Specifically, the application is mainly used for preparing a medicament for treating melanoma, breast cancer, lung cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, stomach cancer, intestinal cancer, head and neck cancer, anal cancer, extrahepatic and biliary tract cancer, bladder cancer, bone cancer, brain stem glioma, brain tumor, bronchial adenoma, burkitt's lymphoma, carcinoid tumor, unknown primary cancer, central nervous system lymph cancer, cervical cancer, children cancer, germ cell tumor, eye cancer, kidney cancer, laryngeal cancer, liver cancer, non-small cell lung cancer, rectal cancer, salivary gland carcinoma, sarcoma, small intestine cancer, soft tissue sarcoma, uterine sarcoma, testicular cancer, leukemia or blood lymphoma.

The invention provides an application of a compound shown in the specification or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of the invention in preparation of a G protein inhibitor.

The invention provides a compound shown in a general formula (I) or a pharmaceutically acceptable salt thereof, or application of a pharmaceutical composition in preparing a medicament for treating tumors. Preferably, the tumor is selected from uveal melanoma, prostate cancer, acute myeloid leukemia, pancreatic cancer or glioblastoma, further preferably uveal melanoma. More preferably, the use is primarily through inhibition of G-proteins.

Compared with the prior art, the invention has the following advantages and effects:

the anti-G protein activity of the tetrahydroimidazo [1,2-a ] pyrazine compound provided by the invention is superior to that of BIM-46174, and the compound has no toxicity to normal cells; and the preparation is simple and the cost is low.

Detailed Description

The present invention will be described in further detail with reference to examples, but the embodiments of the present invention are not limited thereto.

In the present example, the experimental method not specified for the specific conditions is generally performed under the conventional conditions or under the conditions recommended by the manufacturers of the raw materials or the commercial products. Reagents of specific sources are not indicated, and conventional reagents are purchased in the market.

EXAMPLE 1 preparation of target molecules

The thin layer chromatography silica gel plate is HSGF254 of tobacco yellow sea or GF254 of Qingdao, the silica-amine plate used in Thin Layer Chromatography (TLC) is 0.15-0.2 mm, and the thin layer chromatography separation and purification product is 0.4-0.5 mm.

The column chromatography is performed by using 200-mesh and 300-mesh silica gel, and can be purchased from Yangtze river friend silica gel development Co.

The raw materials used in the present invention are mainly available from companies such as Sahn's chemical technology (Shanghai) Co., Ltd, Shanghai Bide medicine science and technology Co., Ltd, national drug group chemical reagent Co., Ltd, and Aladdin chemical reagent Co., Ltd.

In the examples, the solution means an aqueous solution unless otherwise specified.

In the examples, the reaction temperature is, unless otherwise specified, from 20 ℃ to 30 ℃ at room temperature.

The technical scheme adopted by the invention is as follows:

synthetic routes, reagents and conditions for compound (i): a) cs₂CO₃N, N-Dimethylformamide (DMF), room temperature, 86-92%; b) ammonium acetate and toluene, refluxing, 70-85%; c) bromoacetic acid ethyl ester, K₂CO₃，DMF，50℃，90％；d)Pd/C，H₂62-80% of room temperature; e) BH₃THF, reflux, 55-78%; f) DIC, DCM, room temperature, 75-90%; g) triethylamine and DCM at 40 ℃ and 60-85 percent; h) triisopropylsilane, CF₃COOH, room temperature, 80-90%.

Synthesis of Compound 3:

taking compound 1(15.3mmol) and Cs₂CO₃(5.00g, 15.3mmol) in 30mL DMF, mix well and then slowlySlowly adding a compound 2(3.04g,15.3mmol), reacting at room temperature for 20min, after the reaction is finished, evaporating DMF, adding 30mL of methylbenzene for resuspending, taking ammonium acetate (10.6g,138mmol) in a reaction bottle, carrying out reflux reaction for 2h, evaporating methylbenzene, adding a proper amount of water, extracting with ethyl acetate (100mL multiplied by 3), combining ethyl acetate layers, washing with saturated salt water once, drying with anhydrous sodium sulfate, and carrying out reduced pressure evaporation to obtain a compound 3 of a reddish brown oily liquid.

Synthesis of Compound 4:

taking compound 3(28.9mmol) and K₂CO₃(6.02g,43.4mmol) in 50mL DMF, adding ethyl bromoacetate (5.07g,30.3mmol), heating to 50 ℃, reacting for 2 hours, cooling, adding 400mL water, precipitating off-white solid, filtering, drying to obtain off-white solid compound 4, and directly reacting in the next step without purification.

Synthesis of Compound 5:

taking compound 4(7.36mmol) and 10% Pd/C (0.36g) in 15mL of methanol, sealing, and filling with H₂Reacting at room temperature for 8 hours, filtering with diatomite after the reaction is finished to obtain filtrate, concentrating, carrying out silica gel column chromatography, and spin-drying to obtain a white solid compound 5.

Synthesis of Compound 6:

and (2) taking the compound 5(9.70mmol) and 1mol/L borane tetrahydrofuran complex solution (58.2mL,58.2mmol) to be put in 20mL dry tetrahydrofuran, sealing, introducing argon, refluxing for 8 hours, evaporating to remove the tetrahydrofuran after TLC monitoring reaction is finished, adding methanol, refluxing for 2 hours, concentrating, carrying out silica gel column chromatography, and carrying out spin drying to obtain the compound 6 of light yellow oily liquid.

Synthesis of target (I):

taking the compound 7(0.43mmol), adding 2mL of Dichloromethane (DCM) into a reaction bottle, stirring at room temperature, adding Diisopropylcarbodiimide (DIC) (0.08g,0.65mmol) for reaction for 30 minutes, adding the compound 6(0.14g,0.47mmol), reacting at room temperature overnight, monitoring by TLC after the reaction is finished, concentrating, performing silica gel column chromatography, and spin-drying to obtain a white solid. Putting the white solid into a reaction bottle, sealing, introducing argon, adding 1mL of CF₃COOH, stirring at room temperature, adding triisopropylsilane (0.11g,0.47mmol), reacting at room temperature for 2 hours, filtering after the reaction is finished, obtaining filtrate, concentrating, and purifying by semi-preparative liquid phase to obtain the target molecule (I).

Adding the compound 8(0.70mmol) and triethylamine (0.14g,1.41mmol) into 2mL DCM, adding the compound 6(0.21g,0.70mmol), heating to 40 ℃ for overnight reaction, monitoring the reaction by TLC, concentrating, performing silica gel column chromatography, and spin-drying to obtain the target molecule (I).

The target molecule was synthesized according to the above method, and the physicochemical data of the synthesized target molecule were as follows:

compound I-1: the name (S) -3-amino-4- ((S) -8- (cyclohexylmethyl) -2-phenyl-5, 6-dihydroimidazo [1,2-a ] piperazin-7 (8H) -yl) -4-oxobutanenitrile (compound 1 used was (S) -2- (((benzyloxy) carbonyl) amino) -3-cyclohexylpropionic acid, compound 7 was N-tert-butoxycarbonyl-L-asparagine);

¹H NMR(400MHz,CD₃OD):δ8.09(s,1H),7.97–7.86(m,2H),7.50–7.38(m,3H),5.96(t,J＝6.8Hz,1H),4.77–4.71(m,1H),4.60–4.56(m,1H),4.32–4.15(m,2H),3.91–3.85(m,1H),3.01–2.88(m,2H),2.18–2.15(m,1H),1.99–1.96(m,2H),1.64–1.56(m,4H),1.44–0.91(m,6H)；MS(ESI)m/z 392.2[M+H]⁺。

compound I-2: the name (S) -2-amino-1- ((S) -8- (cyclohexylmethyl) -2-phenyl-5, 6-dihydroimidazo [1,2-a ] piperazin-7 (8H) -yl) -3-methylbutan-1-one (compound 1 used was (S) -2- (((benzyloxy) carbonyl) amino) -3-cyclohexylpropionic acid, compound 7 was Boc-L-valine);

¹H NMR(400MHz,CD₃OD)δ7.83(s,1H),7.74–7.70(m,2H),7.53–7.44(m,3H),6.11(dd,J＝10.4,3.6Hz,1H),4.53(d,J＝4.0Hz,1H),4.42(dd,J＝12.8,4.0Hz,2H),4.29–4.22(m,1H),4.06–3.93(m,1H),2.31–2.22(m,2H),2.09–2.02(m,1H),1.82–1.64(m,5H),1.44–1.23(m,4H),1.18(d,J＝6.8Hz,3H),1.10–1.02(m,2H),1.00(d,J＝6.8Hz,3H)；MS(ESI)m/z395.3[M+H]⁺.

compound I-3: the name (S) -2-amino-1- ((S) -8- (cyclohexylmethyl) -2-phenyl-5, 6-dihydroimidazo [1,2-a ] piperazin-7 (8H) -yl) -3- (4-hydroxyphenyl) propan-1-one (compound 1 used was (S) -2- (((benzyloxy) carbonyl) amino) -3-cyclohexylpropionic acid, compound 7 was Boc-O-tert-butyl-L-tyrosine);

¹H NMR(400MHz,CD₃OD)δ7.75–7.68(m,2H),7.59(s,1H),7.55–7.44(m,3H),7.03(d,J＝8.4Hz,2H),6.59(d,J＝8.8Hz,2H),6.06(dd,J＝10.8,3.2Hz,1H),4.74(dd,J＝9.2,6.4Hz,1H),4.20(dd,J＝15.2,4.8Hz,1H),3.93(dd,J＝12.8,4.0Hz,1H),3.87–3.74(m,1H),3.13–3.01(m,2H),2.51–2.44(m,1H),2.30(d,J＝12.4Hz,1H),2.06–1.93(m,1H),1.77–1.61(m,5H),1.45–1.36(m,1H),1.33–1.16(m,3H),1.07–0.98(m,2H)；MS(ESI)m/z 459.3[M+H]⁺。

compound I-4: the name (S) -2-amino-1- ((S) -8- (cyclohexylmethyl) -2-phenyl-5, 6-dihydroimidazo [1,2-a ] piperazin-7 (8H) -yl) -3- (1H-imidazol-4-yl) propan-1-one (compound 1 used was (S) -2- (((benzyloxy) carbonyl) amino) -3-cyclohexylpropionic acid, compound 7 was N-tert-butoxycarbonyl-N' -trityl-L-histidine);

¹H NMR(400MHz,CD₃OD)δ8.87(d,J＝1.2Hz,1H),7.86(s,1H),7.75–7.71(m,2H),7.55–7.44(m,4H),6.14(dd,J＝10.8,3.2Hz,1H),5.05–5.00(m,1H),4.65(dd,J＝15.6,4.0Hz,1H),4.44(dd,J＝13.2,3.6Hz,1H),4.38–4.29(m,1H),4.14–3.99(m,1H),3.47(dd,J＝16.0,4.4Hz,1H),3.30–3.20(m,1H),2.27(d,J＝12.0Hz,1H),2.13–2.06(m,1H),1.83–1.63(m,5H),1.46–1.38(m,1H),1.35–1.19(m,3H),1.08–0.99(m,2H)；MS(ESI)m/z 433.3[M+H]⁺.

compound I-5: the name (S) -2-amino-1- ((S) -8- (cyclohexylmethyl) -2-phenyl-5, 6-dihydroimidazo [1,2-a ] piperazin-7 (8H) -yl) propan-1-one (compound 1 used was (S) -2- (((benzyloxy) carbonyl) amino) -3-cyclohexylpropionic acid, compound 7 was N-tert-butoxycarbonyl-L-alanine);

¹H NMR(400MHz,CD₃OD)δ7.83(s,1H),7.73–7.69(m,2H),7.54–7.44(m,3H),6.09(dd,J＝10.8,2.8Hz,1H),4.64(q,J＝6.8Hz,1H),4.45–4.33(m,2H),4.32–4.23(m,1H),4.04–3.94(m,1H),2.26(d,J＝12.0Hz,1H),2.10–2.03(m,1H),1.80–1.63(m,5H),1.54(d,J＝6.8Hz,3H),1.40–1.20(m,4H),1.10–0.98(m,2H)；MS(ESI)m/z 367.2[M+H]⁺。

compound I-6: the name (S) -2-amino-1- ((S) -8- (cyclohexylmethyl) -2-phenyl-5, 6-dihydroimidazo [1,2-a ] piperazin-7 (8H) -yl) -4-methylpentane-1-one (compound 1 used was (S) -2- (((benzyloxy) carbonyl) amino) -3-cyclohexylpropionic acid, compound 7 was N-tert-butoxycarbonyl-L-leucine);

¹H NMR(400MHz,CD₃OD)δ7.82(s,1H),7.74–7.69(m,2H),7.53–7.43(m,3H),6.09(dd,J＝10.8,3.2Hz,1H),4.66(dd,J＝10.4,3.2Hz,1H),4.49–4.38(m,1H),4.33–4.24(m,2H),4.08–3.98(m,1H),2.25(d,J＝12.0Hz,1H),2.11–2.01(m,1H),1.90–1.57(m,9H),1.47–1.15(m,5H),1.09(d,J＝6.4Hz,3H),1.02(d,J＝6.0Hz,3H)；MS(ESI)m/z 409.2[M+H]⁺。

compound I-7: the name (S) -2-amino-1- ((S) -8- (cyclohexylmethyl) -2-phenyl-5, 6-dihydroimidazo [1,2-a ] piperazin-7 (8H) -yl) -3-phenylpropan-1-one (compound 1 used was (S) -2- (((benzyloxy) carbonyl) amino) -3-cyclohexylpropionic acid, compound 7 was N-tert-butoxycarbonyl-L-phenylalanine);

¹H NMR(400MHz,CD₃OD)δ7.73–7.68(m,2H),7.56(s,1H),7.50(t,J＝6.8Hz,2H),7.44(t,J＝7.2Hz,1H),7.31–7.17(m,5H),6.03(dd,J＝10.8,3.6Hz,1H),4.83–4.79(m,1H),4.16(dd,J＝15.2,4.8Hz,1H),3.94–3.76(m,2H),3.18(d,J＝7.2Hz,2H),2.42–2.35(m,1H),2.28(d,J＝12.8Hz,1H),2.04–1.93(m,1H),1.78–1.64(m,5H),1.42–1.34(m,1H),1.29–1.20(m,3H),1.07–0.98(m,2H)；MS(ESI)m/z 443.3[M+H]⁺。

compound I-8: the name (S) -3-amino-4- ((S) -8- (cyclohexylmethyl) -2-phenyl-5, 6-dihydroimidazo [1,2-a ] piperazin-7 (8H) -yl) -4-oxobutanoic acid (compound 1 used was (S) -2- (((benzyloxy) carbonyl) amino) -3-cyclohexylpropionic acid, compound 7 was tert-butoxycarbonyl-L-aspartic acid-4-tert-butyl ester);

¹H NMR(400MHz,CD₃OD)δ7.81(s,1H),7.73–7.67(m,2H),7.52–7.43(m,3H),6.08(dd,J＝11.2,3.2Hz,1H),4.93–4.92(m,1H),4.48(d,J＝16.4Hz,1H),4.41–4.28(m,2H),4.05–3.97(m,1H),3.05(dd,J＝18.0,6.0Hz,1H),2.87(dd,J＝18.0,7.6Hz,1H),2.25(d,J＝12.4Hz,1H),2.10–2.03(m,1H),1.82–1.66(m,5H),1.46–1.36(m,1H),1.34–1.20(m,3H),1.04(dd,J＝22.4,10.8Hz,2H)；MS(ESI)m/z 411.2[M+H]⁺。

compound I-9: under the name 1- ((S) -4-amino-5- ((S) -8- (cyclohexylmethyl) -2-phenyl-5, 6-dihydroimidazo [1,2-a ] piperazin-7 (8H) -yl) -5-oxopentyl) guanidine (compound 1 used was (S) -2- (((benzyloxy) carbonyl) amino) -3-cyclohexylpropionic acid, compound 7 was tri-tert-butoxycarbonylarginine);

¹H NMR(400MHz,CD₃OD)δ7.84(s,1H),7.74–7.69(m,2H),7.54–7.44(m,3H),6.13(dd,J＝10.8,2.8Hz,1H),4.70(dd,J＝7.6,4.0Hz,1H),4.49–4.37(m,2H),4.30–4.25(m,1H),4.08–3.95(m,1H),3.25(t,J＝6.8Hz,2H),2.26(d,J＝12.0Hz,1H),2.11–1.96(m,2H),1.93–1.84(m,1H),1.81–1.61(m,7H),1.41–1.19(m,4H),1.08–0.95(m,2H)；MS(ESI)m/z 452.3[M+H]⁺。

compound I-10: the name (2S,3R) -2-amino-1- ((S) -8- (cyclohexylmethyl) -2-phenyl-5, 6-dihydroimidazo [1,2-a ] piperazin-7 (8H) -yl) -3-methylpentane-1-one (compound 1 used was (S) -2- (((benzyloxy) carbonyl) amino) -3-cyclohexylpropionic acid, compound 7 was N-tert-butoxycarbonyl-L-isoleucine);

¹H NMR(400MHz,CD₃OD)δ7.80(s,1H),7.71(d,J＝6.8Hz,2H),7.52–7.43(m,3H),6.09(dd,J＝10.0,3.6Hz,1H),4.53(d,J＝4.0Hz,1H),4.44–4.38(m,2H),4.24–4.14(m,1H),4.06–3.95(m,1H),2.24(d,J＝12.0Hz,1H),2.07–1.95(m,2H),1.82–1.65(m,5H),1.57–1.51(m,1H),1.46–1.38(m,1H),1.36–1.19(m,4H),1.16(d,J＝7.2Hz,3H),1.08–1.00(m,2H),0.94(t,J＝7.6Hz,3H)；MS(ESI)m/z 409.3[M+H]⁺。

compound I-11: the name (S) -4-amino-5- ((S) -8- (cyclohexylmethyl) -2-phenyl-5, 6-dihydroimidazo [1,2-a ] piperazin-7 (8H) -yl) -5-oxopentanoic acid (the compound 1(S) -2- (((benzyloxy) carbonyl) amino) -3-cyclohexylpropionic acid used, compound 7 is N-tert-butoxycarbonyl-L-glutamic acid-5-tert-butyl ester);

¹H NMR(400MHz,CD₃OD)δ7.85(s,1H),7.76–7.67(m,2H),7.56–7.43(m,3H),6.06(dd,J＝11.6,3.2Hz,1H),4.74(dd,J＝7.2,3.6Hz,1H),4.64–4.46(m,2H),4.40(dd,J＝12.8,4.0Hz,1H),4.09–3.94(m,1H),2.68–2.53(m,2H),2.26(d,J＝12.4Hz,1H),2.20–2.00(m,3H),1.81–1.61(m,5H),1.42–1.19(m,4H),1.04(dd,J＝23.6,12.0Hz,2H)；MS(ESI)m/z425.3[M+H]⁺.

compound I-12: the name (S) -3-amino-4- ((S) -8- (cyclohexylmethyl) -2-phenyl-5, 6-dihydroimidazo [1,2-a ] piperazin-7 (8H) -yl) -4-oxobutanamide (compound 1 used was (S) -2- (((benzyloxy) carbonyl) amino) -3-cyclohexylpropionic acid, compound 7 was tert-butoxycarbonyl-N-beta-trityl-L-asparagine);

¹H NMR(400MHz,CD₃OD)δ7.84(s,1H),7.74–7.69(m,2H),7.54–7.44(m,3H),6.12(dd,J＝10.8,2.4Hz,1H),4.87–4.75(m,1H),4.50(d,J＝15.6Hz,1H),4.40–4.33(m,2H),4.06–3.93(m,1H),2.99(dd,J＝16.4,6.0Hz,1H),2.80(dd,J＝16.8,7.2Hz,1H),2.27(d,J＝12.0Hz,1H),2.13–2.01(m,1H),1.81–1.63(m,5H),1.45–1.35(m,1H),1.34–1.17(m,3H),1.04(dd,J＝23.6,11.6Hz,2H)；MS(ESI)m/z 410.3[M+H]⁺。

compound I-13: the name (2S,3R) -2-amino-1- ((S) -8- (cyclohexylmethyl) -2-phenyl-5, 6-dihydroimidazo [1,2-a ] piperazin-7 (8H) -yl) -3-hydroxybutan-1-one (compound 1 used was (S) -2- ((((benzyloxy) carbonyl) amino) -3-cyclohexylpropionic acid, compound 7 was N-tert-butoxycarbonyl-O-tert-butyl-L-threonine);

¹H NMR(400MHz,CD₃OD)δ7.83(s,1H),7.74–7.69(m,2H),7.54–7.44(m,3H),6.14(d,J＝8.4Hz,1H),4.64(d,J＝3.2Hz,1H),4.50(d,J＝14.8Hz,1H),4.42–4.31(m,2H),4.27–4.21(m,1H),4.07–3.86(m,1H),2.27(d,J＝11.6Hz,1H),2.11–2.03(m,1H),1.81–1.64(m,5H),1.40–1.20(m,4H),1.30(d,J＝6.0Hz,3H),1.08–0.99(m,2H)；MS(ESI)m/z 397.3[M+H]⁺。

compound I-14: the name (S) -2, 6-diamino-1- ((S) -8- (cyclohexylmethyl) -2-phenyl-5, 6-dihydroimidazo [1,2-a ] piperazin-7 (8H) -yl) hexan-1-one (compound 1 used was (S) -2- (((benzyloxy) carbonyl) amino) -3-cyclohexylpropionic acid, compound 7 was (S) -2, 6-di-tert-butoxycarbonylaminocaproic acid);

¹H NMR(400MHz,CD₃OD)δ7.82(s,1H),7.75–7.69(m,2H),7.55–7.42(m,3H),6.09(dd,J＝10.8,3.2Hz,1H),4.66(dd,J＝7.6,4.0Hz,1H),4.45–4.38(m,2H),4.30–4.20(m,1H),4.09–3.94(m,1H),2.93(t,J＝8.0Hz,2H),2.25(d,J＝12.0Hz,1H),2.06–2.02(m,1H),1.97–1.84(m,2H),1.81–1.63(m,7H),1.59–1.48(m,2H),1.40–1.19(m,4H),1.08–0.99(m,2H)；MS(ESI)m/z 424.3[M+H]⁺。

compound I-15: the name (S) -4-amino-5- ((S) -8- (cyclohexylmethyl) -2-phenyl-5, 6-dihydroimidazo [1,2-a ] piperazin-7 (8H) -yl) -5-oxopentanamide (compound 1 used was (S) -2- (((benzyloxy) carbonyl) amino) -3-cyclohexylpropionic acid, compound 7 was N-tert-butoxycarbonyl-N' -trityl-L-glutamine);

¹H NMR(400MHz,CD₃OD)δ7.85(d,J＝4.8Hz,1H),7.74–7.68(m,2H),7.54–7.45(m,3H),6.19–6.04(m,1H),4.88–4.84(m,1H),4.72–4.55(m,1H),4.49–4.33(m,2H),4.07–3.88(m,1H),2.56–2.52(m,1H),2.44–2.32(m,1H),2.31–2.15(m,2H),2.15–1.99(m,2H),1.80–1.61(m,5H),1.39–1.18(m,4H),1.03(dd,J＝22.0,10.0Hz,2H)；MS(ESI)m/z 424.3[M+H]⁺。

compound I-16: the title compound is (S) -2-amino-1- ((S) -8- (cyclohexylmethyl) -2-phenyl-5, 6-dihydroimidazo [1,2-a ] piperazin-7 (8H) -yl) -3- (1H-indol-3-yl) propan-1-one (compound 1 used is (S) -2- (((benzyloxy) carbonyl) amino) -3-cyclohexylpropionic acid, compound 7 is N-tert-butoxycarbonyl-N' -tert-butoxycarbonyl-L-tryptophan, 144599-95-1).

¹H NMR(400MHz,CD₃OD)δ7.69–7.59(m,2H),7.57–7.48(m,3H),7.37(d,J＝7.2Hz,1H),7.29(s,1H),7.23(s,1H),7.19(dd,J＝7.2,1.6Hz,1H),6.78–6.71(m,2H),5.82(dd,J＝11.2,3.2Hz,1H),4.76(dd,J＝9.6,6.0Hz,1H),4.14(dd,J＝14.8,5.2Hz,1H),3.72–3.58(m,2H),3.46–3.33(m,2H),2.33(d,J＝12.0Hz,1H),1.93–1.72(m,3H),1.69(d,J＝9.6Hz,2H),1.62–1.52(m,2H),1.39–1.21(m,4H),1.04–0.95(m,2H)；MS(ESI)m/z 482.3[M+H]⁺.

Compound I-17: the name was (S) -2-amino-3-cyclohexyl-1- ((S) -8- (cyclohexylmethyl) -2-phenyl-5, 6-dihydroimidazo [1,2-a ] piperazin-7 (8H) -yl) propan-1-one (compound 1 used was (S) -2- (((benzyloxy) carbonyl) amino) -3-cyclohexylpropionic acid, compound 7 was (S) -2- ((tert-butoxycarbonyl) amino) -3-cyclohexylpropionic acid).

¹H NMR(400MHz,CD₃OD)δ7.83(s,1H),7.75–7.69(m,2H),7.52–7.41(m,3H),6.06(dd,J＝10.8,3.2Hz,1H),4.65(dd,J＝10.4,3.2Hz,1H),4.50–4.38(m,1H),4.31–4.21(m,2H),4.09–3.97(m,1H),2.11–2.01(m,4H),1.90–1.57(m,10H),1.53–1.15(m,4H)1.38–1.20(m,4H),1.06–0.96(m,4H)；MS(ESI)m/z 449.3[M+H]⁺。

Compound I-18: the name was (S) -1- ((S) -8- (cyclohexylmethyl) -2-phenyl-5, 6-dihydroimidazo [1,2-a ] piperazin-7 (8H) -yl) -3-mercapto-2-methylaminopropane-1-one (compound 1 used was (S) -2- (((benzyloxy) carbonyl) amino) -3-cyclohexylpropionic acid, compound 7 was N-Boc-N-methyl-S-trityl-L-cysteine, 91292-54-5).

¹H NMR(400MHz,CD₃OD)δ7.85(s,1H),7.76–7.69(m,2H),7.54–7.44(m,3H),6.20(dd,J＝11.2,3.2Hz,1H),4.83(t,J＝5.6Hz,1H),4.50(dd,J＝15.2,4.0Hz,1H),4.45–4.32(m,2H),4.09–3.97(m,1H),3.13(dd,J＝15.2,6.0Hz,1H),3.07(dd,J＝14.8,5.2Hz,1H),2.77(s,3H),2.30(d,J＝12.0Hz,1H),2.16–2.05(m,1H),1.86–1.64(m,5H),1.34–1.18(m,4H),1.12–1.01(m,2H)；MS(ESI)m/z 413.2[M+H]⁺.

Compound I-19: the name was (S) -1- ((S) -8- (cyclohexylmethyl) -2-phenyl-5, 6-dihydroimidazo [1,2-a ] piperazin-7 (8H) -yl) -2- (dimethylamino) -3-mercaptopropan-1-one (compound 1 used was (S) -2- (((benzyloxy) carbonyl) amino) -3-cyclohexylpropionic acid, compound 7 was N, N-dimethyl-S-trityl-L-cysteine, 1275955-00-4).

¹H NMR(400MHz,CD₃OD)δ7.82(s,1H),7.73–7.69(m,2H),7.53–7.43(m,3H),6.20(dd,J＝11.6,3.2Hz,1H),4.89–4.86(m,1H),4.55(d,J＝15.6Hz,1H),4.42–4.37(m,2H),4.11–3.98(m,1H),3.21–3.12(m,2H),2.98(s,6H),2.30(d,J＝12.0Hz,1H),2.17–2.05(m,1H),1.86–1.67(m,5H),1.31–1.18(m,4H),1.12–1.03(m,2H)；MS(ESI)m/z 427.3[M+H]⁺。

Compound I-20: the name was (S) -1- ((S) -8- (cyclohexylmethyl) -2-phenyl-5, 6-dihydroimidazo [1,2-a ] piperazin-7 (8H) -yl) -2- (diethylamino) -3-mercaptopropan-1-one (compound 1 used was (S) -2- (((benzyloxy) carbonyl) amino) -3-cyclohexylpropionic acid, compound 7 was N, N-diethyl-S-trityl-L-cysteine). The preparation of compound 7 was as follows: the compound S-trityl-L-cysteine (cas number 2799-07-7) (2.75mmol) was taken and dissolved in 10mL of methanol at 0 ℃ with stirring, acetaldehyde (6.87mmol) was slowly added, reaction was carried out at 0 ℃ for 10min, then sodium borohydride (4.13mmol) was added, and reaction was carried out at 0 ℃ for 2 hours. After the reaction, the methanol is distilled off, and white solid N, N-diethyl-S-trityl-L-cysteine is obtained by silica gel column chromatography.

¹H NMR(400MHz,CD₃OD)δ7.83(s,1H),7.75–7.68(m,2H),7.54–7.44(m,3H),6.24(dd,J＝11.6,2.8Hz,1H),4.96–4.91(m,1H),4.67(dd,J＝15.2,4.0Hz,1H),4.49–4.39(m,2H),4.14–4.00(m,1H),3.46–3.37(m,2H),3.28–3.23(m,2H),3.20(dd,J＝14.4,3.6Hz,1H),3.10(dd,J＝14.8,8.0Hz,1H),2.32(d,J＝12.4Hz,1H),2.19–2.09(m,1H),1.87–1.63(m,5H),1.38(t,J＝7.6Hz,6H),1.30–1.17(m,4H),1.13–1.03(m,2H)；MS(ESI)m/z 455.3[M+H]⁺.

Compound I-21: the name was (S) -1- (8- (cyclohexylmethyl) -2-phenyl-5, 6-dihydroimidazo [1,2-a ] piperazin-7 (8H) -yl) -2-methylpropan-1-one (compound 1(S) -2- (((benzyloxy) carbonyl) amino) -3-cyclohexylpropionic acid and compound 7 was isobutyric acid used).

¹H NMR(400MHz,CD₃OD)δ7.84(s,1H),7.71(d,J＝7.2Hz,2H),7.57–7.40(m,3H),6.22(dd,J＝11.6,2.8Hz,1H),4.50(dd,J＝15.2,3.6Hz,1H),4.34(dd,J＝12.8,3.6Hz,1H),4.28–4.20(m,1H),3.97–3.81(m,1H),3.15–3.05(m,1H),2.17(d,J＝12.8Hz,1H),2.09–2.00(m,1H),1.80–1.61(m,5H),1.37–1.22(m,4H),1.20(d,J＝6.8Hz,3H),1.16(d,J＝6.4Hz,3H),1.09–0.97(m,2H)；MS(ESI)m/z 366.3[M+H]⁺.

Compound I-22: the name 1- ((S) -8- (cyclohexylmethyl) -2-phenyl-5, 6-dihydroimidazo [1,2-a ] piperazin-7 (8H) -yl) -2-methylbutan-1-one (compound 1 used was (S) -2- (((benzyloxy) carbonyl) amino) -3-cyclohexylpropionic acid, compound 7 was (S) -2-methylbutanoic acid).

¹H NMR(400MHz,CD₃OD)δ7.83(s,1H),7.70(d,J＝6.8Hz,2H),7.54–7.44(m,3H),6.24(dd,J＝11.6,3.2Hz,1H),4.54(dd,J＝15.2,4.0Hz,1H),4.35(dd,J＝13.2,3.6Hz,1H),4.22–4.14(m,1H),3.97–3.80(m,1H),2.96–2.85(m,1H),2.21(d,J＝11.6Hz,1H),2.10–2.00(m,1H),1.80–1.62(m,6H),1.52–1.43(m,1H),1.32–1.22(m,3H),1.20(d,J＝7.2Hz,3H),1.14–0.97(m,3H),0.92(t,J＝7.2Hz,3H)；MS(ESI)m/z 380.3[M+H]⁺.

Compound I-23: the name is (2S,3R) -2-amino-1- ((S) -8-benzyl-2-phenyl-5, 6-dihydroimidazo [1,2-a ] piperazin-7 (8H) -yl) -3-methylpentane-1-one (compound 1 used is N-benzyloxycarbonyl-L-phenylalanine, compound 7 is N-tert-butoxycarbonyl-L-isoleucine);

¹H NMR(400MHz,CD₃OD):δ8.07(s,1H),7.97–7.86(m,2H),7.50–7.38(m,3H),7.30–7.23(m,3H),7.19(d,J＝7.8Hz,2H),5.96(t,J＝6.8Hz,1H),4.23–4.13(m,2H),4.06–3.96(m,2H),3.38(d,J＝6.8Hz,1H),3.01–2.88(m,2H),1.88–1.77(m,1H),1.16(d,J＝7.2Hz,3H),1.10–1.00(m,2H),0.95(t,J＝7.6Hz,3H)；MS(ESI)m/z 403.2[M+H]⁺。

compound I-24: the name (S) -2-amino-1- ((R) -8- (cyclohexylmethyl) -2-phenyl-5, 6-dihydroimidazo [1,2-a ] piperazin-7 (8H) -yl) -3- (4-hydroxyphenyl) propan-1-one (compound 1 used was (R) -2- (((benzyloxy) carbonyl) amino) -3-cyclohexylpropionic acid, compound 7 was Boc-O-tert-butyl-L-tyrosine, 154802-74-1);

¹H NMR(400MHz,CD₃OD)δ7.78(s,1H),7.72–7.67(m,2H),7.51–7.42(m,3H),7.20(d,J＝8.4Hz,2H),6.82(d,J＝8.4Hz,2H),6.14(dd,J＝11.6,3.2Hz,1H),4.79(dd,J＝8.8,6.4Hz,1H),4.30–4.17(m,2H),3.93(dd,J＝14.8,3.6Hz,1H),3.47–3.38(m,1H),3.19–3.06(m,2H),2.25(d,J＝12.0Hz,1H),1.93–1.85(m,1H),1.81–1.71(m,3H),1.68–1.61(m,2H),1.40–1.34(m,1H),1.31–1.22(m,3H),1.10–1.01(m,2H)；MS(ESI)m/z 459.3[M+H]⁺。

compound I-25: the name is (S) -2-amino-1- ((R) -8-isobutyl-2-phenyl-5, 6-dihydroimidazo [1,2-a ] piperazin-7 (8H) -yl) -3-mercaptopropane-1-one (compound 1 used is N-benzyloxycarbonyl-D-leucine and compound 7 is N-tert-butoxycarbonyl-S-trityl-L-cysteine);

¹H NMR(400MHz,CD₃OD)δ7.87(s,1H),7.74–7.68(m,2H),7.55–7.44(m,3H),6.18(dd,J＝11.6,2.4Hz,1H),4.77(dd,J＝7.2,5.6Hz,1H),4.51–4.42(m,1H),4.37–4.33(m,2H),4.04–3.92(m,1H),3.18(dd,J＝14.8,5.6Hz,1H),3.00(dd,J＝14.8,7.2Hz,1H),2.21–2.13(m,1H),1.78–1.61(m,2H),1.12(d,J＝6.4Hz,3H),0.99(d,J＝6.4Hz,3H)；MS(ESI)m/z 359.2[M+H]⁺。

compound I-26: the name is (S) -2-amino-1- ((S) -8- ((R) -sec-butyl) -2-phenyl-5, 6-dihydroimidazo [1,2-a ] piperazin-7 (8H) -yl) -3-mercaptopropan-1-one (compound 1 used is N-benzyloxycarbonyl-L-isoleucine, compound 7 is N-tert-butoxycarbonyl-S-trityl-L-cysteine);

¹H NMR(400MHz,CD₃OD)δ7.88(s,1H),7.72(d,J＝6.8Hz,2H),7.55–7.45(m,3H),5.81(d,J＝7.6Hz,1H),4.76(dd,J＝7.2,5.2Hz,1H),4.54–4.42(m,2H),4.40–4.31(m,1H),4.09–3.98(m,1H),3.08(dd,J＝14.8,5.2Hz,1H),2.90(dd,J＝14.8,7.2Hz,1H),2.27–2.19(m,1H),1.59–1.52(m,1H),1.34–1.26(m,1H),1.15(d,J＝6.8Hz,3H),0.96(t,J＝7.6Hz,3H)；MS(ESI)m/z 359.2[M+H]⁺.

compound I-27: the name is (S) -2-amino-1- ((S) -8-isopropyl-2-phenyl-5, 6-dihydroimidazo [1,2-a ] piperazin-7 (8H) -yl) -3-mercaptopropane-1-one (compound 1 used is N-benzyloxycarbonyl-L-valine, compound 7 is N-tert-butoxycarbonyl-S-trityl-L-cysteine).

¹H NMR(400MHz,CD₃OD)δ7.88(s,1H),7.73(d,J＝7.2Hz,2H),7.54–7.45(m,3H),5.76(d,J＝8.0Hz,1H),4.78(dd,J＝7.2,5.2Hz,1H),4.56–4.43(m,2H),4.40–4.30(m,1H),4.10–3.98(m,1H),3.09(dd,J＝14.8,5.2Hz,1H),2.91(dd,J＝14.8,7.2Hz,1H),2.52–2.43(m,1H),1.20(d,J＝6.8Hz,3H),1.06(d,J＝6.8Hz,3H)；MS(ESI)m/z 345.2[M+H]⁺.

Compound I-28: the name is (S) -2-amino-3-mercapto-1- ((S) -8-methyl-2-phenyl-5, 6-dihydroimidazo [1,2-a ] piperazin-7 (8H) -yl) propan-1-one (compound 1 used is N-benzyloxycarbonyl-L-alanine, compound 7 is N-tert-butoxycarbonyl-S-trityl-L-cysteine).

¹H NMR(400MHz,CD₃OD)δ7.87(s,1H),7.74–7.70(m,2H),7.54–7.44(m,3H),5.91(q,J＝6.8Hz,1H),4.74–4.67(m,1H),4.52–4.29(m,3H),4.01–3.87(m,1H),3.09(dd,J＝14.8,5.6Hz,1H),2.94(dd,J＝14.8,7.2Hz,1H),1.69(d,J＝6.8Hz,3H)；MS(ESI)m/z 317.1[M+H]⁺。

Compound I-29: the name is (S) -8- (cyclohexylmethyl) -7- (isopropylsulfonyl) -2-phenyl-5, 6,7, 8-tetrahydroimidazo [1,2-a ] piperazine (compound 1 used is (S) -2- (((benzyloxy) carbonyl) amino) -3-cyclohexylpropionic acid, compound 8 is isopropylsulfonyl chloride).

¹H NMR(400MHz,CD₃OD):δ8.12(s,1H),7.98–7.85(m,2H),7.56–7.43(m,3H),5.99(t,J＝6.8Hz,1H),4.62–4.54(m,1H),4.35–4.16(m,2H),3.92–3.85(m,1H),3.32–3.26(m,1H),2.18–2.15(m,1H),2.00–1.97(m,2H),1.66–1.56(m,4H),1.46–0.95(m,6H),1.31(d,J＝7.2Hz,6H)；MS(ESI)m/z 402.2[M+H]⁺.

Compound I-30: the name was (S) -2-amino-1- (8- (cyclohexylmethyl) -2-phenyl-5, 6-dihydroimidazo [1,2-a ] piperazin-7 (8H) -yl) ethan-1-one (compound 1 used was (S) -2- (((benzyloxy) carbonyl) amino) -3-cyclohexylpropionic acid, compound 7 was N-tert-butoxycarbonylglycine).

¹H NMR(400MHz,CD₃OD)δ7.84(s,1H),7.74–7.68(m,2H),7.53–7.44(m,3H),6.13(dd,J＝11.6,2.8Hz,1H),4.33(d,J＝5.6Hz,2H),4.27–4.10(m,3H),3.99–3.86(m,1H),2.25(d,J＝12.4Hz,1H),2.12–2.02(m,1H),1.81–1.62(m,5H),1.41–1.32(m,1H),1.31–1.19(m,3H),1.11–1.02(m,2H)；MS(ESI)m/z 353.2[M+H]⁺.

Compound I-31: the title compound was (S) -8- (cyclohexylmethyl) -7- (ethylsulfonyl) -2-phenyl-5, 6,7, 8-tetrahydroimidazo [1,2-a ] piperazine (compound 1 used was (S) -2- (((benzyloxy) carbonyl) amino) -3-cyclohexylpropionic acid, compound 8 was ethylsulfonyl chloride).

¹H NMR(400MHz,CD₃OD):δ8.07(s,1H),7.96–7.85(m,2H),7.58–7.43(m,3H),5.96(t,J＝6.8Hz,1H),4.63–4.53(m,1H),4.35–4.18(m,2H),3.90–3.83(m,1H),3.49–3.36(m,2H),2.16–2.12(m,1H),2.00–1.95(m,2H),1.67–1.56(m,4H),1.46–0.94(m,6H),1.20(t,J＝7.2Hz,3H)；MS(ESI)m/z 388.2[M+H]⁺.

Compound I-32: the name is (S) -8-benzyl-7- (isopropylsulfonyl) -2-phenyl-5, 6,7, 8-tetrahydroimidazo [1,2-a ] piperazine (compound 1 used is N-benzyloxycarbonyl-L-phenylalanine, compound 8 is isopropylsulfonyl chloride);

¹H NMR(400MHz,CD₃OD):δ8.09(s,1H),7.96–7.87(m,2H),7.51–7.39(m,3H),7.31–7.24(m,3H),7.18(d,J＝7.8Hz,2H),5.98(t,J＝6.8Hz,1H),4.61–4.55(m,1H),4.34–4.16(m,2H),3.92–3.85(m,1H),3.36(d,J＝7.8Hz,1H),3.31–3.18(m,1H),3.03(d,J＝7.8Hz,1H),1.35(d,J＝7.2Hz,6H)；MS(ESI)m/z 396.2[M+H]⁺。

compound I-33: the name (S) -2, 6-diamino-1- ((R) -8-isobutyl-2-phenyl-5, 6-dihydroimidazo [1,2-a ] piperazin-7 (8H) -yl) hexan-1-one (compound 1 used was N-benzyloxycarbonyl-D-leucine and compound 7 was (S) -2, 6-di-tert-butoxycarbonylaminocaproic acid);

¹H NMR(400MHz,CD₃OD)δ7.85(s,1H),7.76–7.68(m,2H),7.54–7.43(m,3H),6.18(dd,J＝11.6,2.8Hz,1H),4.65(dd,J＝8.0,4.8Hz,1H),4.39–4.31(m,3H),4.03–3.94(m,1H),2.97(t,J＝8.0Hz,2H),2.22–2.09(m,1H),2.06–1.84(m,2H),1.82–1.68(m,3H),1.65–1.51(m,3H),1.12(d,J＝6.4Hz,3H),1.00(d,J＝6.8Hz,3H)；MS(ESI)m/z 384.3[M+H]⁺。

compound I-34: the compound is named as (S) -1- ((S) -8- (cyclohexylmethyl) -2-phenyl-5, 6-dihydroimidazo [1,2-a ] piperazin-7 (8H) -yl) -2-hydroxypropan-1-one (the compound 1 used is (S) -2- (((benzyloxy) carbonyl) amino) -3-cyclohexylpropionic acid, the compound 7 is O-tert-butoxycarbonyl-L-lactic acid, the cas number is 2042339-34-2, and the compound is named as (2S) -2- [ [ (1, 1-dimethyllethoxy) carbonyl ] oxy ] propanoic acid);

¹H NMR(400MHz,CD₃OD)δ7.82(s,1H),7.74–7.67(m,2H),7.55–7.44(m,3H),6.12(d,J＝9.6Hz,1H),4.79–4.71(m,1H),4.64(dd,J＝12.8,6.4Hz,1H),4.34–4.24(m,2H),3.91–3.78(m,1H),2.24(d,J＝12.0Hz,1H),2.11–2.01(m,1H),1.80–1.63(m,5H),1.42(d,J＝6.4Hz,3H),1.36–1.18(m,4H),1.10–0.99(m,2H)；MS(ESI)m/z 368.2[M+H]⁺.

compound I-35: the name (R) -1- (8-isobutyl-2-phenyl-5, 6-dihydroimidazo [1,2-a ] piperazin-7 (8H) -yl) -2-methylpropan-1-one (compound 1 used is N-benzyloxycarbonyl-D-leucine and compound 7 is isobutyric acid);

¹H NMR(400MHz,CD₃OD)δ7.84(s,1H),7.71(d,J＝6.8Hz,2H),7.53–7.44(m,3H),6.20(dd,J＝11.6,2.4Hz,1H),4.50(dd,J＝15.2,3.6Hz,1H),4.35(dd,J＝13.2,3.6Hz,1H),4.28–4.21(m,1H),3.97–3.84(m,1H),3.15–3.02(m,1H),2.17–2.06(m,1H),1.75–1.66(m,1H),1.65–1.50(m,1H),1.19(d,J＝6.8Hz,3H),1.16(d,J＝6.4Hz,3H),1.12(d,J＝6.4Hz,3H),0.97(d,J＝6.4Hz,3H)；MS(ESI)m/z 326.2[M+H]⁺.

compound I-36: the name (2S,3R) -2-amino-3-methyl-1- ((R) -8-methyl-2-phenyl-5, 6-dihydroimidazo [1,2-a ] piperazin-7 (8H) -yl) pentan-1-one (compound 1 used is N-benzyloxycarbonyl-D-alanine and compound 7 is N-tert-butoxycarbonyl-L-isoleucine);

¹H NMR(400MHz,CD₃OD)δ7.81(s,1H),7.72(d,J＝6.8Hz,2H),7.51–7.41(m,3H),6.02(q,J＝6.8Hz,1H),4.23–4.13(m,1H),4.06–3.96(m,1H),3.38(d,J＝6.8Hz,1H),3.07(dd,J＝14.8,5.6Hz,1H),2.95(dd,J＝14.8,7.2Hz,1H),1.88–1.77(m,1H),1.48(d,J＝7.2Hz,3H),1.16(d,J＝7.2Hz,3H),1.10–1.00(m,2H),0.95(t,J＝7.6Hz,3H)；MS(ESI)m/z 327.2[M+H]⁺。

method for salifying target molecule

The preparation method of the inorganic acid salt comprises the following steps: dissolving a target molecule (1mmol) in 10mL of anhydrous methanol, slowly dropwise adding 5mL of anhydrous methanol solution containing inorganic acid (1mmol) under ice bath, stirring for 30 minutes at the temperature after dropwise adding, and then evaporating methanol at normal temperature to obtain the inorganic acid salt of the target molecule. By the method, hydrochloride (I-10-1), hydrobromide (I-10-2), sulfate (I-10-3) and methanesulfonate (I-10-4) of the compound I-10 are prepared;

the preparation method of the organic acid salt comprises the following steps: dissolving a target molecule (1mmol) in 10mL of anhydrous methanol, slowly dropwise adding 5mL of dry ether solution containing organic acid (1mmol) in ice bath, stirring for 30 minutes at the temperature after dropwise adding, and then evaporating the solvent at normal temperature to obtain the organic acid salt of the target molecule. By this method, maleate salt (I-10-5), succinate salt (I-10-6) and fumarate salt (I-10-7) of compound I-10 were prepared.

Preparation of a mixture of two target molecules

And (3) putting the two target molecules with equal molar weight (1mmol) into anhydrous methanol (5mL), stirring for 10 minutes at room temperature, and evaporating the solvent at room temperature to obtain a mixture of the target molecules. Three target molecule mixtures of (I-3) - (I-10), (I-10) - (I-21) and (I-3) - (I-21) were prepared by this method.

Example 2 evaluation of biological Activity of target molecule

1. Method for testing Gq protein inhibitory activity in vitro

Detection was performed using IP-ONE-Gq KIT (cat # 62 IPAPEEB (1,000 tests)):

1) preparation of IP1 detection reagent

Stimulation Buffer (Stimulation Buffer): 1 volume of 5 XStimB mother liquor was diluted with 4 volumes of distilled water for further use.

Detection Reagent Solutions (Detection Reagent Solutions): 1 volume of 6 Xdetection reagent stock solution was diluted with 5 volumes of Lysis Buffer (lysine & Detection Buffer) for use.

2) Standard curve for IP1

First, IP1 standard was prepared as shown in Table 1 and was used.

TABLE 1 preparation of IP1 Standard

② a 384-well plate is taken, 14 mu L of IP1 standard substance is added into each well of the experimental group according to Std7-Std0, 14 mu L of StimB is added into each well of the blank group, and each group has three multiple wells.

③ 3. mu.L of IP1 d2 Reagent solution was added to each well of the experimental group, and 3. mu.L of lysis buffer was added to each well of the blank group.

And fourthly, adding 3 mu L of IP1 Tb Crytate Antibody solution into each hole of the experimental group and the blank group.

And covering the cover, and incubating for 1 hour at room temperature.

And sixthly, detecting by an HTRF reader sample introduction.

And (c) drawing a standard curve of the IP 1.

3) Determination of IC of Gq protein inhibitor₅₀Value of

7 μ L of CHO-M1 cells (which stably express muscarinic receptor 1 in CHO cells (M1) disclosed in the literature "Total Synthesis and Structure-activity relationship study of a series of selective Gq protein inhibitors, Nature chemistry.2016,8, 1035-1041") were added per well according to the Cisbio kit instructions. Each group of three multiple holes.

② 7 microliter StimB is added into each hole of the blank group and the control group, and 7 microliter StimB containing 2 multiplied by the compound to be tested is added into each hole of the experimental group.

③ centrifuging at 600 Xg for 1 minute, mixing well, and incubating at 37 ℃ for 1 hour.

Fourthly, 3 mu L of lysis buffer is added into each hole of the blank group, and 3 mu L of IP1 d2 Reagent solution is added into each hole of the control group and the experimental group.

Fifthly, adding 3 mu L of IP1 Tb Cryptate Antibody solution into each hole.

Sixthly, incubating for 1 hour at room temperature.

And seventhly, detecting the sample injection of the HTRF reader.

The results of analysis are shown in Table 4.

2. Method for testing inhibitory activity of Gi protein in vitro

Detection was performed using CAMP-Gi KIT (cat # 62AM9PEB (1,000 tests)):

1) preparation of cAMP detection reagent

Detection Reagent Solutions (Detection Reagent Solutions): 1 volume of the 5 XcAMP Eu Detection reagent mother liquor is taken and diluted with 4 volumes of Lysis Buffer (lysine & Detection Buffer) for use. 1 volume of 5 XcAMP-d 2 antibody stock was diluted with 4 volumes of lysis buffer for use.

2) Preparation of a Standard Curve for cAMP

Preparation of cAMP Standard products as shown in Table 2.

TABLE 2 preparation of cAMP standards

② a 384-well plate is taken, 5 mu L cAMP standard substance is added into each hole of the experimental group according to Std7-Std0, 5 mu L StimB is added into each hole of the blank group, and each group has three multiple holes.

③ 5. mu.L of StimB was added to each well of the experimental and blank groups.

And fourthly, adding 5 mu L of cAMP Eu detection reagent solution into each hole of the experimental group and the blank group.

Fifthly, 5 mu L of cAMP-d2 antibody solution is added into each hole of the experimental group, and 5 mu L of lysis buffer solution is added into each hole of the blank group.

Sixthly, the cover is covered, and the mixture is incubated for 1 hour at room temperature.

And seventhly, detecting the sample injection of the HTRF reader.

And drawing a cAMP standard curve.

3) Determination of IC of Gi protein inhibitor₅₀Value of

Add 5. mu.L of a resuspension of CHO-mGlu2 cells (purchased from Girale Biotech, Inc., and CHO cells stably expressing metabotropic glutamate receptor 2) containing 3-isobutyl-1-methylxanthine (IBXM) per well according to Cisbio kit instructions. Each group of three multiple holes.

② 4 microliter StimB is added into each hole of the blank group and the control group, and 4 microliter StimB containing 2.5 multiplied by the compound to be tested is added into each hole of the experimental group.

③ centrifuging at 600 Xg for 1 minute, mixing well, and incubating at 37 ℃ for a suitable time.

Separately, 1 μ L of StimB was added to each well of the blank group, and 1 μ L of forskolin solution with a concentration of 15 μ M was added to each well of the control group and the experimental group.

Centrifuging at 600 Xg for 1 min, mixing, and incubating at 37 deg.C for a proper time.

Sixthly, adding 5 mu L of cAMP Eu detection reagent solution into each hole.

Seventhly, 5 mu L of lysis buffer is added to each hole of the blank group, and 5 mu L of cAMP-d2 antibody solution is added to each hole of the control group and the experimental group.

And incubating at room temperature for 1 hour.

And ninthly, HTRF reader sampling detection.

Analysis of results in r is shown in table 4.

3. In vitro Gs protein inhibitory activity test method

The detection was carried out using CAMP-GS DYNAMIC KIT ((cat # 62AM4PEB (1,000 tests)):

1) preparation of cAMP detection reagent

2) Preparation of a Standard Curve for cAMP

First, cAMP standards were prepared as shown in Table 3 and were used.

TABLE 3 preparation of cAMP standards

mu.L of cAMP-d2 antibody solution is added to each well of the experimental group, and 5. mu.L of lysis buffer is added to each well of the blank group.

Fifthly, adding 5 mu L of cAMP Eu detection reagent solution into each hole of the experimental group and the blank group.

And seventhly, detecting the sample injection of the HTRF reader.

And drawing a cAMP standard curve.

3) Determination of IC of Gs protein inhibitors₅₀Value of

[ 5 μ L of IBXM-containing HEK293- β 2AR cells (a strain of HEK293 cells stably expressing β 2 adrenergic receptors, disclosed in the literature "Total Synthesis and Structure-activity relationship among students of a series of selective Gq protein inhibitors, Nature chemistry.2016,8, 1035-. Each group of three multiple holes.

② 5 microliter StimB is added into each hole of the blank group and the control group, and 5 microliter StimB containing 2 multiplied by the compound to be tested is added into each hole of the experimental group.

5 mu L of lysis buffer is added into each hole of the blank group, and 5 mu L of cAMP-d2 antibody solution is added into each hole of the control group and the experimental group.

Fifthly, 5 mu L of cAMP Eu detection reagent solution is added into each hole.

Sixthly, centrifuging for 1 minute at 600 Xg, mixing evenly, and incubating for a proper time at room temperature.

And seventhly, detecting the sample injection of the HTRF reader.

The results of analysis are shown in Table 4.

4. Cell growth experiment (CCK-8 detection method)

Cell inoculation: cells in logarithmic growth phase were collected and cell suspension concentration was adjusted to 5X 10 per well³Each cell, 100 μ L per well volume, was seeded into 96-well plates, each set of 4 replicates (marginal wells filled with sterile PBS);

cell culture: after cell attachment, 0% FBS-containing RPMI-1640 medium was starved for 8 hours, and the control group was cultured in 10% (v/v) FBS-containing RPMI-1640 complete medium at 37 ℃ with 5% CO₂Continuously culturing in an incubator (culturing for different times according to experimental requirements);

color generation: adding 10 μ L CCK-8 solution (5mg/mL) into the three groups of cells after culturing for 72 hours, terminating the culture within 4 hours, and oscillating on a shaking table at low speed for 10min to fully dissolve crystals;

color comparison: the absorbance (OD) of each well was measured on an ELISA detector, and the absorbance of each well was measured by selecting a wavelength of 450nm, using a cell-free RPMl-1640 culture solution well as a blank group, and using a cell well without stimulation by a compound as a control group. The experiment was repeated three times.

The results are recorded: the inhibition rate of cell growth is ═ x 100% (absorbance value of control group-absorbance value of experimental group)/(absorbance value of control group-absorbance value of blank group) ], and the proliferation rate of cell is ═ x 100% (absorbance value of experimental group-absorbance value of blank group)/(absorbance value of control group-absorbance of blank group) ];

drawing a cell growth curve: the cell growth curve was plotted with time as abscissa and inhibition/proliferation rate as ordinate.

Gra in GraphPad softwareInhibitor concentrations were plotted in phPad Prism mapping software to determine log [ inhibitor [ ]]Variable slope model estimation of IC versus response₅₀。

The test results are shown in table 4, and table 4 shows the effect of the obtained compounds on the inhibition of the activity of G protein and the proliferation of antitumor cells.

TABLE 4

a:IC₅₀: half the effective inhibitory concentration;

b:92.1 (available from Shanghai Rich-balance Biotech, Inc.), MP41 is a typical uveal melanoma cell (available from American ATCC cell Bank), human prostate cancer cell LNCaP (available from American ATCC cell Bank), and human acute promyelocytic leukemia cell HL60 (available from American ATCC cell Bank).

c Stable expression of M₁CHO normal cells of the recipient.

The biological activity results show that the molecule has stronger inhibition effect on the G protein, and the effective inhibition concentration IC of most compounds₅₀The value is better than BIM-46174. The results of anti-cell proliferation activity revealed that some of the compounds had very potent inhibitory effects on uveal melanoma cells (92.1 and MP41), with compounds I-3, I-10, I-16 and I-30 also showing unexpected superior activity to BIM-46174. In addition, BIM-46174 has strong toxicity, especially has obvious killing effect on normal cells, and does not have druggability, but the compound in the invention has no obvious killing effect on normal cells, which indicates that the compound has higher safety, indicates that the molecules have the potential of developing novel high-efficiency G protein inhibitors, and has larger potential for treating related tumor diseases, especially uveal melanoma, prostatic cancer, acute myeloid leukemia, pancreatic cancer or glioblastomaThe application value of (2).

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Claims

1. A tetrahydroimidazo [1,2-a ] pyrazine compound or a pharmaceutically acceptable salt thereof, characterized in that: the chemical structure of the tetrahydroimidazo [1,2-a ] pyrazine compound is shown in a formula (I):

wherein X is selected from carbonyl; l is selected from-CH-or hydrogen;

R₁selected from hydrogen or methyl;

R₂selected from cyano groups,

R₃Selected from methyl, amino, hydroxyl,

R₄Selected from methyl, isopropyl, and isopropyl,

2. A tetrahydroimidazo [1,2-a ] pyrazine compound or a pharmaceutically acceptable salt thereof, characterized in that: the tetrahydroimidazo [1,2-a ] pyrazine compound is a compound with the following name:

3. The tetrahydroimidazo [1,2-a ] pyrazine compound according to claim 1 or 2, or pharmaceutically acceptable salts thereof, characterized in that:

the pharmaceutically acceptable salt is obtained by treating with inorganic acid and organic acid.

4. The tetrahydroimidazo [1,2-a ] pyrazine compound according to claim 3, or pharmaceutically acceptable salts thereof, characterized in that:

the pharmaceutically acceptable salt is sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, chloride, bromide, iodide, acetate, caprate, caprylate, acrylate, formate, isobutyrate, hexanoate, heptanoate, propionate, oxalate, malonate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, gamma-hydroxybutyrate, glycolate, tartrate, mandelate and sulfonate.

5. A process for the preparation of tetrahydroimidazo [1,2-a ] pyrazines according to any one of claims 1 to 4, or a pharmaceutically acceptable salt thereof, comprising the steps of:

R₄selected from methyl, isopropyl, and isopropyl,

(3) dispersing compound 4 and catalyst D in organic solvent D, sealing, and charging H₂Reacting, filtering, concentrating the obtained filtrate, and purifying to obtain a compound 5; the catalyst D is Pd/C with the dry content of 10 percent;

(A) dissolving the compound 7 in an organic solvent G, and adding diisopropylcarbodiimide to carry out a first reaction; then adding a compound 6, carrying out a second reaction, and after the TLC monitoring reaction is finished, concentrating and purifying; adding CF to the obtained product under inert atmosphere₃COOH and triisopropylsilane, and a third reaction to obtain the tetrahydroimidazo [1,2-a ] according to any one of claims 1 to 4]Pyrazine compounds;

(B) dissolving a compound 8 and triethylamine in an organic solvent H, adding a compound 6, reacting, monitoring by TLC, concentrating and purifying to obtain the tetrahydroimidazo [1,2-a ] pyrazine compound according to any one of claims 1-4;

the compound 7 in the step (A) is N-tert-butyloxycarbonyl-L-asparagine, Boc-O-tert-butyl-L-tyrosine, N-tert-butyloxycarbonyl-N '-trityl-L-histidine, N-tert-butyloxycarbonyl-L-phenylalanine, tert-butyloxycarbonyl-L-aspartic acid-4-tert-butyl ester, tri-tert-butyloxycarbonyl-arginine, N-tert-butyloxycarbonyl-L-isoleucine, N-tert-butyloxycarbonyl-L-glutamic acid-5-tert-butyl ester, tert-butyloxycarbonyl-N-beta-trityl-L-asparagine, (S) -2, 6-di-tert-butyloxycarbonyl-aminocaproic acid, N-tert-butyloxycarbonyl-N' -trityl-L-glutamine, N-tert-butyloxycarbonyl-L-histidine, N-butyloxycarbonyl-L-histidine, N-tert-butyloxycarbonyl-L-histidine, N-butyloxycarbonyl-L-histidine, N-methyl-L-glutamate, N-L-histidine, N-tert-butyloxycarbonyl-L-histidine, N, t-butyloxycarbonyl, N, t-tert-butyloxycarbonyl, N, t-butyloxycarbonyl, N, t-tert-butyloxycarbonyl, N, t-tert-butyloxycarbonyl, N, N-tert-butoxycarbonyl-N' -tert-butoxycarbonyl-L-tryptophan, (S) -2- ((tert-butoxycarbonyl) amino) -3-cyclohexylpropionic acid, isobutyric acid, (S) -2-methylbutyric acid, N-tert-butoxycarbonylglycine, (S) -2, 6-di-tert-butoxycarbonylaminocaproic acid or O-tert-butoxycarbonyl-L-lactic acid;

the compound 8 in the step (B) is isopropyl sulfonyl chloride or ethyl sulfonyl chloride.

6. A pharmaceutical composition having G protein inhibitory activity, characterized by: contains active ingredients or contains active ingredients and a drug carrier; the active ingredient comprises at least one of the tetrahydroimidazo [1,2-a ] pyrazines compound or the pharmaceutically acceptable salt thereof according to any one of claims 1 to 4.

7. The pharmaceutical composition having G protein inhibitory activity according to claim 6, wherein: the active ingredient may also include one or more other pharmaceutically active compounds.

8. Use of the tetrahydroimidazo [1,2-a ] pyrazine compound according to any one of claims 1 to 4 or pharmaceutically acceptable salt thereof, or pharmaceutical composition according to claim 6 or 7 for preparing a G protein inhibitor.

9. Use of the tetrahydroimidazo [1,2-a ] pyrazine compound according to any one of claims 1 to 4 or pharmaceutically acceptable salt thereof and the pharmaceutical composition according to claim 6 or 7 for preparing a medicament for treating tumors.

10. Use according to claim 9, characterized in that: the tumor is melanoma, breast cancer, lung cancer, pancreatic cancer, ovarian cancer, prostatic cancer, gastric cancer, intestinal cancer, head and neck cancer, anal cancer, cancer of the extrahepatic and biliary tract, bladder cancer, bone cancer, brain stem glioma, brain tumor, bronchial adenoma, Burkitt's lymphoma, carcinoid tumor, unknown primary cancer, lymph cancer of the central nervous system, cervical cancer, cancer of children, germ cell tumor, eye cancer, renal cancer, laryngeal carcinoma, liver cancer, non-small cell lung cancer, rectal cancer, salivary gland carcinoma, sarcoma, small intestine cancer, soft tissue sarcoma, uterine sarcoma, testicular cancer, leukemia or blood lymphoma.